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14 protocols using azure 600

1

Protein Expression Analysis of hiPSC-Derived CD34+ Cells

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Magnetically sorted hiPSC-derived CD34+ cells were washed three times with cold PBS, lysed in protein-preservative buffers, and stepwise prepared for polyacrylamide gel electrophoresis, subsequently transferred to membranes by electroblotting as previously described [30 (link)]. Afterward, membranes were incubated with the primary antibodies: p-YAP1 (phospho-Ser127, 1:500, Abcam), YAP1 (1:1000, Abcam), p-WWTR1 (phospho-Ser89, 1:500, Invitrogen), WWTR1 (1:1000, Invitrogen), and GAPDH (1:20,000, Invitrogen), overnight at 4 °C. Subsequently, membranes incubated with antibodies were washed five times with Tris-buffered saline plus Tween 20 and incubated with HRP-conjugated secondary antibodies. Targeted signals were exposed with HRP Substrate and detected by Azure 600 (Azure Biosystems). Detailed information on these antibodies and reagents is provided in Additional file 1: Table S1.
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2

Baicalein-Induced DNA Fragmentation by Copper Ions

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The ability of baicalein to induce DNA breakage either alone or in the presence of copper ions was measured by determining the conversion level of the supercoiled form of plasmid DNA to open-circular and linear forms. The final, total reaction volume of 25 µL: 2 µL (1 µg) of pBR322 plasmid DNA (Thermo Scientific) in 10 mM Tris–HCl buffer (pH 7.5) was incubated with baicalein and/or 100 μM CuSO4 for 1 h at 37 °C. For control reactions, the plasmid DNA was incubated without the tested compound but in the presence of copper ions or DMSO (solvent control). The baicalein stock solution was prepared each time anew; the compound was suspended in DMSO and heated at 65 °C to dissolve before use. DNA fragmentation was analyzed by performing gel electrophoresis. After incubation, samples were mixed with 5 µL of gel loading buffer and loaded on 1% agarose gel. The gels were stained with SimplySafe (EURX) and photographed using a UV light in Azure 600 (Azure Biosystems, Dublin, CA, USA).
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3

Protein Expression Analysis in Transfected Cells

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Total protein was extracted from transfected cells using a lysis buffer containing 1% phenylmethyl sulfonyl fluoride, and the concentration of total protein was determined using a protein concentration assay Kit (Pierce, Thermo Fisher Scientific Inc.). Protein samples were separated using 10% SDS–PAGE gel at a rate of 20 µg per well and then transferred to polyvinylidene difluoride membranes. The membranes were washed with 1 × TBST and incubated with the following primary antibodies: anti–DGAT1 (ab100982; Abcam, Cambridge, UK), anti–DGAT2 (ab237613; Abcam), anti–peroxisome proliferator-activated receptor γ (PPARγ) (bs-4509R; Bioss, Beijing, China), anti–CCAAT/enhancer-binding protein α (C/EBPα) (bs-1630R; Bioss), anti–C/EBPβ (bs-1396R; Bioss), and anti-lipin 1(LPIN1) (bs-7533R; Bioss) overnight at 4 °C under constant shaking. This was followed by incubation with the secondary antibody: horseradish peroxidase-conjugated affinipure goat anti-mouse IgG (Jackson, Anhui, China) for 2 h at room temperature (approximately 25 °C) after washing with PBS. The endogenous control was β-Actin (AB8226; Abcam). Protein bands were photographed using a multifunctional imaging system (Azure 600; Azure Biosystems, Dublin, CA, USA).
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4

Protein Fractionation and Western Blot Analysis

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The whole cell lysates were prepared by using RIPA Lysis and Extraction Buffer (Thermo Scientific, IL, USA) with Pierce Protease and Phosphatase Inhibitor Mini (Thermo Scientific, IL, USA). The fractionated lysates were prepared by using the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Scientific, IL, USA). Protein concentrations were measured using the Bio-Rad Protein Assay (Bio-Rad; CA, USA), and the concentration in individual samples was equalized before adding 4x Laemmli buffer to a final concentration of 1x. Equal amounts of protein (50 μg whole cell lysates, 45 μg fractionated lysates) were run on 10% SDS-PAGE gels and then transferred with iBlot 2 Transfer Stacks and iBlot 2 Dry Blotting System onto PVDF membranes (Invitrogen, Thermo Scientific, IL, USA). After one hour 5% milk block, membranes were probed with the antibodies diluted with 5% BSA (NRF3) or 5% milk (β-actin and PCNA and secondary antibodies) in Tris-buffered saline with 0.1% Tween 20. Primary antibodies anti-NRF3 (HPA055889, Sigma-Aldrich), anti-β-actin (NB600-501, Novus Biologicals, UK), and anti-PCNA (NB600-501SS, Novus Biologicals, UK) were incubated overnight, and appropriate HRP-conjugated secondary antibodies (sc-2054 and sc-2055, Santa Cruz, CA, USA) were incubated at RT for one hour. Blots were detected with the Western blot imaging system Azure 600 (Azure Biosystems, CA, USA).
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5

Poly(A) Tail-Preserving RNA Sequencing Protocol

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The experimental procedure was based on the protocol by Sallés and Strickland (1999) [46 (link)], with the following modifications. To keep the entire length of the polyA tail of each transcript, the total RNA was extracted by the phenol-chloroform method. Isolated RNA was incubated with 1 µL of 50 μM oligo-dT per sample for 5 min at 65 °C. The ligation mix was prepared from the following components: T4 ligase (1 µL), Superscript IV 5× buffer (5 µL), 20U/µL RNAse inhibitor (1 µL), 10 mM dNTP (1 µL), 10mM ATP (1 µL), 1M MgCl2 (0.1 µL), 0,1M DTT (2 µL), and RNAse-free water (2 µL). Each sample was processed in 10 µL of the mixture and incubated for 30 min at 42 °C to allow T4-mediated oligo-dT ligation. Anchoring was carried out by incubating with 1 µL of oligo-dT anchor (5′-GCGAGCTCCGCGGCCGCGT-3′) for 1 h at 12 °C, followed by 2 min of incubation at 42 °C. cDNA synthesis was performed by the addition of 1 µL of Superscript II Reverse Transcriptase with the following setup: 45 min at 45 °C, 10 min at 80 °C, hold at 4 °C. Prepared cDNA was subjected to PCR with gene-specific forward primers and anchoring reverse primer (Supplementary Table S1). Images were developed in an Azure 600 (Azure Biosystems). Each experiment was carried out in technical triplicates.
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6

Quantification of HSP-70 Expression in hMPCs

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Protein lysates from hMPCs were isolated with the RIPA buffer supplemented with a 0.1% protease inhibitor cocktail 48 h after the external stimulus. The cell extract (which contained 20 μg of protein) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After blotting onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA, USA), the membranes were probed with HSP-70 and β-actin, followed by incubation with HRP-conjugated secondary antibodies. Immunoblots were visualized using a chemiluminescence imaging system (Azure 600, Azure biosystems, Dublin, CA, USA) with enhanced chemiluminescence reagent. With the use of ImageJ, the relative intensity of HSP-70 was quantified and compared with the expression of β-actin, which was then normalized by the value derived from hMPCs that were cultured on a flat surface without EF stimulus (Flat).
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7

Protein Characterization via Analytical Techniques

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Purified proteins (1 μg) were used to qualitatively determine the isoelectric point using Novex IEF gels (Thermofisher, Waltham, MA, Cat# EC6655BOX) as described by the manufacturer. Purity and protein stability were determined using SDS-PAGE (Any Kda Protein Gel, Bio Rad, Hercules, CA, Cat# 4569036), 1 μg protein, and EZBlue Coomassie stain (Sigma Aldrich, St. Louis, MO, Cat# G1041-500 ML) as described by the manufacturer. Immunoblotting was performed using up to 60 μg of cell lysate, 10 μL of harvest media, or 200 ng of purified protein. Using Azure 600 (Azure Biosystems, Dublin, CA), the proteins were imaged using the following primary and secondary antibodies: TAG-72 (Santa Cruz biotech, Santa Cruz, CA, Cat# sc-20042), beta actin (Cell Signaling Technology, Danvers, MA, Cat# 4970 and 3700), ST6GalNAc 1 and C3GNT (Proteintech, Rosemont, IL, Cat# 15363-1-AP and 21291-1-AP), anti-human Fc (Novus Biologicals, Centennial, CO, Cat# NBP1-40876), and several near infrared secondary antibodies (Licor, Bad Homburg, Germany, Cat# 926-68072, 926-68-021, and 926-32232).
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8

Quantifying Lung Antioxidant Proteins

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Total lung homogenates were prepared in RIPA assay buffer and analyzed
by Western blotting using the following antibodies: goat polyclonal human SOD3
antibody, goat polyclonal mouse SOD3 antibody (both 1:500; R&D Systems,
Minneapolis, MN), and mouse monoclonal β-actin antibody (1:10000; Sigma).
Donkey anti-goat HRP (IgG H&L) antibody was used at 1:2000 for mouse and
human SOD3, and donkey anti-mouse HRP (IgG H&L) antibody was used at 1:10000
for β-actin (both Santa Cruz Biotechnology, Dallas, TX). Membranes were
developed using Radiance ECL chemiluminescent HRP substrate per product protocol
(Azure Biosystems, Dublin, CA) and imaged on the Azure 600 (Azure
Biosystems).
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9

Western Blot Analysis of LCN2/NGAL in RPE/Choroid

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Protein was extracted from the RPE/choroid, according to a previously described methodology [28 (link)] and protein concentrations were measured by Pierce™ BCA protein assay kit (Thermo Fisher Scientific). Subsequently, 10 μg of protein was loaded into each well, separated on a 4–12% Bis-Tris SDS-PAGE gel (NuPAGE, Invitrogen), and transferred to a polyvinylidene fluoride membrane (Bio-Rad). After blocking with 5% bovine serum albumin (BSA, Sigma) in Tris-buffered saline (TBS, Bio-Rad), membranes were incubated overnight with primary antibodies: anti-mouse LCN2/NGAL (1:1000; catalog# AF1857, R&D Systems), or anti-α-Tubulin (1:1000; catalog# T6199, Millipore Sigma), used as a loading control. After three washes with 0.1% Tween-20 in TBS, incubated for 1 h with appropriate secondary antibody, either horseradish peroxidase (HRP)-conjugated anti-goat IgG (1:7500; Jackson ImmunoResearch), or HRP-conjugated anti-mouse IgG (1:8000; GE healthcare Lifesciences). Membranes were developed using SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific). Images were acquired using an Azure c500 or Azure 600 gel imager with cSeries Capture Software (Azure Biosystems).
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10

Western Blot Analysis of Protein Expression

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Total proteins were extracted by RIPA buffer (Beyotime, China) containing 1nmol/L PMSF (Beyotime). The proteins were resolved by SDS-PAGE and then transferred on a polyvinylidene difluoride membrane (GE Healthcare Life Sciences, USA). After incubation with 5% skimmed milk at room temperature for 2 h, the blot was hybridized with the primary antibody at 4 °C overnight. After washing by TBST three times, the blot was incubated with the secondary antibody (Beyotime, A0208, A0216, dilution: 1:1000) at room temperature for 1 h. After adding the enhanced chemiluminescence (NCM Biotech, China), the immunoblots were obtained using Azure600 (azure biosystems, USA). The primary antibodies used in this study were GAPDH (Abcam, UK, ab181602, dilution: 1:5000), TDP-43 (Abcam, ab190963, dilution: 1:2000), ABHD2 (absin, China, abs140798, dilution: 1:1000), Cleaved-caspase3 (Cell Signaling, USA, #9664, dilution: 1:2000), p53 (Proteintech, USA, 10442-1-AP, dilution: 1:5000), Bax (Cell Signaling, #5023, dilution: 1:1000), Bcl-2(Abcam, ab182858, dilution: 1:2000), Ki67 (Abcam, ab16667, dilution: 1:1000).
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